During the development of immature CD4-CD8- (double negative or DN) thymocytes to the CD4-CD8-(double positive or DP) stage, thymocytes attempt to construct the antigen binding subunits of the T cell receptor (TCR) by recombining individual gene segments, first those of TCRB and then those of TCRa, into a single coding unit. The fidelity with which the error-prone DNA rearrangements occur is quality controlled according to a checkpoint termed B-selection. B-selection stipulates that only thymocytes which maintain the translational reading frame of TCRB during B rearrangement will be permitted to mature to the DP stage. "Inframe" B-rearrangements trigger thymocyte maturation through the activation of a developmental precursor of the TCR called the pre-TCR complex, whose hallmark is an invariant 33kD subunit termed pre-Ta (pTa). While most cell surface receptor complexes are activated by ligand engagement, for which antibody (Ab) stimulation frequently serves as an effective surrogate, this does not appear to be true for the pre-TCR complex. Ab engagement of the pre-TCR in vivo fails to promote maturation of thymocytes to the DP stage (Hunig, 1988; Takahama et a!., 1995). Moreover, removal of the potential ligand-binding exodomains of TCRB and pTa does not abrogate the ability of the pre-TCR to promote thymocyte development (Irving et al., 1998). Thus, it is clear from transgenic and gene-targeting experiments that production of a functional TCRB protein product is required to induce pre-TCR signal transduction; however, our understanding of how pre-TCR signals are induced and then linked to the subsequent differentiation program is rudimentary. The goal of this proposal is to gain insight into how early thymocyte development is controlled by the preTCR complex. We will do so by completion of the following aims: i) to investigate the molecular basis for and physiologic significance of a novel pre-TCR isoform; ii) to assess the role of pTa in initiation of pre-TCR signaling; and iii) to determine the manner in which pre-TCR signals are linked to the DNA-binding proteins responsible for executing the ensuing differentiation program. The proposed experiments should provide insight into how pre-TCR signals control thymocyte development as well as how aberrant pre-TCR signals can lead to malignant transformation.